The long-term objectives and specific aims of this research plan are to characterize two recently identified trans-acting regulatory proteins involved in the induction of expression of Herpes simplex virus 1 alpha (immediate early) genes. Previous work has shown that these proteins (70 and 77 kd) modulate the activity of a virion protein, the alpha-trans-inducing factor (alpha-TIF). Alpha-TIF is responsible for the induction of alpha gene expression by a mechanism which requires the presence of a specific viral cis-acting DNA sequence and at least one host cell nuclear factor. In Part I of this study, antibody directed against the two proteins will be obtained by the use of synthetic peptides, bacterial expression systems or the use of in vitro transcribed and translated m-RNAs encoding the two proteins. Mutant viruses, which fail to express the genes, or which express truncated proteins will be constructed and used in Part II of the study to characterize the functions of the 70 and 77 kd proteins in vivo. If either of the genes are essential, mutants ts in these functions will be constructed or cell lines which express the HSV-1 genes or their varicella zoster virus homologs will be constructed to complement the viral deletion mutants in a host range system. The localization of the proteins will be determined in vivo by indirect immunofluorescence and/or pulse-labeling followed by immunochemical analysis. Part III of the study will include the purification of the two proteins by antibody affinity or conventional chromatography. The purified proteins will be used to characterize their functions in vitro. These studies will include the identification of complex formation by in vitro crosslinking and DNA binding assays. Elucidation of the functions of the 70 and 77 kd proteins in alpha gene regulation is significant for the following reasons: i) additional information on the mechanisms responsible for the regulation of HSV-1 gene expression will enhance the methods of controlling or preventing HSV-1 infections, ii) additional information on viral-host cell interactions is necessary to further understand the mechanisms by which these pathogens establish dominance over the host cell synthetic machinery, and iii) it would provide a model system in which to study viral-cell interactions at the transcriptional level.